No abstract

The Streptococcusfaecalis plasmid pCF-10 is representative of a class of plasmids that enables its host cells to respond to sex pheromones produced by other S. faecalis cells. The pheromone response has been previously shown to result in increased conjugal plasmid transfer, cell clumping, and multiple cell-surface antigemic changes. To test for other effects of pheromone induction, cells carrying pCF-10 were used as recipients in matings with an isogenic donor strain carrying a derivative of pCF.10, tagged with a transposon to provide an additional selective marker. Pheromone induction of the "male recipients" decreased their recipient ability by a factor of 10-300 in comparison to uninduced cells or plasmidfree recipients. These results indicate that an entry exclusion (surface exclusion) function, similar to that described in studies of plasmids in Gram-negative bacteria, is induced during the S. faecalis pheromone response process. The exclusion operates only against homologous plasmids. Immunological, biochemical, and genetic experiments using monoclonal antibodies reactive with C130, the predominant protein antigen associated with the pheromone response of cells carrying pCF-10, indicate that this antigen is involved in surface exclusion. The data also support the notion that synthesis of C130 involves a posttranslational modification of a precursor of C130 to a fmal product of higher molecular weight form.Conjugal transfer of certain Streptococcusfaecalis plasmids is enhanced as a result of the response of donor cells carrying these plasmids to peptide pheromones (clumping inducing agents) produced by recipients (1, 2). The pheromone response of cells carrying the tetracycline-resistance plasmid, pCF-10 (3), involves the synthesis of multiple cell-surface antigens (4, 5). Presumably, one result of these antigenic changes is to increase the ability of donor cells to attach to recipients via synthesis of an adhesin termed "aggregation substance" (2). This system represents an attractive bacterial model for the analysis of cell-hormone interactions, and it also provides an opportunity to combine genetic, biochemical, and immunological techniques to analyze the streptococcal cell surface at the molecular level.Our previous immunoblotting studies (4) indicated that the predominant surface antigenic change associated with the pheromone response is the synthesis of proteinaceous antigenic material that migrates as a group of bands in the 130-kDa range in denaturing polyacrylamide gels. We have termed this group of bands the C130 antigen. A second antigen, SA73, that migrates as a single band of 73 kDa, also appears during pheromone induction. We have obtained monoclonal antibodies that react with at least two bands in the C130 group (5). Preliminary experiments with these antibodies indicated that C130 was not involved in the processes of attachment or transfer of DNA between donor and recipient cells.Conjugative plasmids in Gram-negative bacteria confer, by at least two different mechanisms, immunity to ...

A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the "BAC TG-EMBED" method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.